Lsm 700 confocal system

Manufactured by Zeiss

Sourced in Germany

The LSM 700 is a confocal microscopy system designed by Zeiss. It is used for high-resolution imaging of biological specimens. The system utilizes laser excitation and pinhole optics to capture sharp, in-focus images from selected focal planes within a sample.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions) Wga texas red x, by Thermo Fisher Scientific (3 mentions) Cardiomyocyte immunocytochemistry kit, by Thermo Fisher Scientific (2 mentions) Anti sarcomeric α actinin antibody, by Merck Group (2 mentions) Alexa fluor 488 conjugated anti mouse, by Thermo Fisher Scientific (2 mentions) Planapo 40x 1.2 oil objective, by Zeiss (2 mentions) Eclipse ti e microscope, by Nikon (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions) Nis elements software, by Nikon (2 mentions) Zen software, by Zeiss (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 conjugated anti rabbit, by Jackson ImmunoResearch (1 mentions) Alexa 647, by Jackson ImmunoResearch (1 mentions) Mouse anti repo 8d12, by Developmental Studies Hybridoma Bank (1 mentions) Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions) Alexa 488, by Jackson ImmunoResearch (1 mentions) Tcs nt confocal system, by Leica (1 mentions) Optosplit, by Cairn Research (1 mentions) Alexa fluor 594 conjugated anti rabbit, by Thermo Fisher Scientific (1 mentions)

32 protocols using lsm 700 confocal system

Immunostaining of Cardiomyocyte Sarcomeres

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Cells were prepared for staining using Cardiomyocyte Immunocytochemistry Kit (Thermo Fisher Scientific) following manufacturer’s instructions. Sarcomeres were stained using anti-α-sarcomeric actinin antibody (Sigma; 1:500, mouse) overnight at 4 °C. The day after, samples were incubated for 1 h at room temperature with Alexa Fluor 488 conjugated anti-mouse (Life Technologies, 1:250) and phalloidin TRITC conjugated (Sigma-Aldrich, 1:500) for actin staining. DAPI was used to stain nuclei (Invitrogen). Images were collected using Zeiss LSM 700 confocal system equipped with a PlanApo 40x/1.2 oil objective at 2048 × 2048 pixels per image with a 100 Hz acquisition rate, and analyzed as previously described [61 (link)].

Di Sante M., Antonucci S., Pontarollo L., Cappellaro I., Segat F., Deshwal S., Greotti E., Grilo L.F., Menabò R., Di Lisa F, & Kaludercic N. (2023). Monoamine oxidase A-dependent ROS formation modulates human cardiomyocyte differentiation through AKT and WNT activation. Basic Research in Cardiology, 118(1), 4.

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Quantifying LDLR Expression by Immunofluorescence

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Samples were fixed in 4% paraformaldehyde followed by permeabilization using 0.1% saponin. Cells were then incubated with anti-LDLR (1:100 dilution; BioVision) overnight. Alexa Fluor 488-conjugated anti-rabbit (1:200 dilution; 715–546–150 Jackson ImmunoResearch, Pennsylvania) was used for visualization. DAPI (ThermoFischer Scientific) was used to stain DNA. All pictures were taken with a Zeiss LSM 700 confocal system.

Bjune K., Wierød L, & Naderi S. (2018). Triciribine increases LDLR expression and LDL uptake through stabilization of LDLR mRNA. Scientific Reports, 8, 16174.

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Drosophila Larval Brain Dissection and Imaging

Cited 2 times

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Larval brains were dissected with forceps, fixed in 4% paraformaldehyde, processed, stained, and imaged as previously described (11 (link)). The following antibodies were used: 8D12 mouse anti-Repo (1:10, Developmental Studies Hybridoma Bank), anti-phospho-S21-Sqh (1:500, gift of Robert Ward) (26 (link)), anti-Anillin (1:500, gift of Maria Giansanti) (27 (link)). Secondary antibodies were conjugated to Cy3 (1:150), Alexa-488, or Alexa-647 (1:100) (Jackson Laboratories). Brains were mounted on glass slides ventral side down in vectashield and whole mount imaged on a Zeiss LSM 700 confocal system. For experiments where protein levels were compared between genotypes, all samples were prepared, subjected to immunohistochemistry, imaged, and image processed in a parallel manner side by side. 6 or more brains were stained with each Ab combination, and representative images are shown for each result. All brain phenotypes shown were highly penetrant, with approximately 75-100% of animals showing the growth phenotypes described. Images were analyzed in Zeiss Zen Software and processed in Photoshop. Larval Drosophila brain hemisphere volumes were analyzed using Imaris software. Larval glial cells were counted manually in representative optical sections of age-matched brain hemispheres, matched for section plane. Statistical analyses were done using Prism.

Chen A.S., Wardwell-Ozgo J., Shah N.N., Wright D., Appin C.L., Vigneswaran K., Brat D.J., Kornblum H.I, & Read R.D. (2018). Drak/STK17A drives neoplastic glial proliferation through modulation of MRLC signaling. Cancer research, 79(6), 1085-1097.

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Quantifying Synaptic Markers in Neuronal Cultures

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Confocal images of the neuronal cell cultures were captured using an LSM700 confocal system (Zeiss). The original images were analyzed using ImageJ (NIH) (https://imagej.nih.gov/ij/index.html). The number and size (area) of the synapse marker-positive puncta defined with proper thresholding (top 2.5–5.0% of the display range) were quantified. The morphologies of the neurons were visualized by staining with the neuronal marker MAP2. Nuclei were visualized with 4′,6-diamidino-2-phenylindole stains.
Quantification in the artificial synapse formation assay was performed as previously described (35 (link), 36 (link)). In brief, after the appropriate threshold had been set, the immunoreactive areas on the surface of the HEK293T cells were measured. Approximately 20 to 40 cells from more than 10 separate fields per culture were quantified per group. The morphologies of the HEK293T cells were visualized by coexpressing GFP.

Darwish M., Ito M., Iijima Y., Takase A., Ayukawa N., Suzuki S., Tanaka M., Komori K., Kaida D, & Iijima T. (2023). Neuronal SAM68 differentially regulates alternative last exon splicing and ensures proper synapse development and function. The Journal of Biological Chemistry, 299(10), 105168.

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Live Cell Imaging of Signaling

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Cells were washed once with ice-cold PBS, fixed in 4% ice-cold formaldehyde in PBS and afterwards washed twice with PBS. Upon addition of calyculin A and 8-CPT-cAMP, live imaging was employed. The confocal images were obtained with a Zeiss LSM700 confocal system with a 63× oil objective lens.

Burgers P.P., Bruystens J., Burnley R.J., Nikolaev V.O., Keshwani M., Wu J., Janssen B.J., Taylor S.S., Heck A.J, & Scholten A. (2016). Structure of smAKAP and its regulation by PKA-mediated phosphorylation. The FEBS journal, 283(11), 2132-2148.

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Live-cell Imaging of Calcium Dynamics

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Cell lines used for imaging include HEK293 and HeLa cells. All cells were grown in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10 mM HEPES and 10% heat-inactivated fetal bovine serum, unless otherwise noted. Transfections were performed using Lipofectamine 2000 (Life Technologies) following the manufacturer's protocol. To aid efficient and stable puncta formation, the DMEM medium was substituted by pre-warmed Ca²⁺-free Hank's Balanced Salt Solution (HBSS) before imaging. 0.5–1 μM thapsigargin (TG) or 2.5 μM ionomycin was used to induce store depletion. Live cell imaging was performed at room temperature with 60× oil lens on an inverted Nikon Eclipse Ti-E microscope customized with A1R-A1 confocal and motorized total internal reflection fluorescence (TIRF) modules by using argon-ion (405 nm and 488 nm) and helium-neon (543 nm) or diode (561 nm) as laser sources. In some experiments, a Zeiss LSM 700 confocal system equipped with a 100× oil lens (NA = 1.45) was used to obtain images with higher resolution. Image analysis was performed using the NIS-Elements software (Nikon) or Image J (NIH).

Ma G., Wei M., He L., Liu C., Wu B., Zhang S.L., Jing J., Liang X., Senes A., Tan P., Li S., Sun A., Bi Y., Zhong L., Si H., Shen Y., Li M., Lee M.S., Zhou W., Wang J., Wang Y, & Zhou Y. (2015). Inside-out Ca2+ signalling prompted by STIM1 conformational switch. Nature Communications, 6, 7826.

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Live-cell imaging of Ca2+ signaling

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Cell lines used for imaging include HEK293 and HeLa cells. All cells were grown in DMEM (Sigma) supplemented with 10 mM HEPES and 10% heat-inactivated fetal bovine serum, unless otherwise noted. Transfections were performed using Lipofectamine 2000 (Life Technologies) following the manufacturer's protocol. To aid efficient and stable puncta formation, the DMEM medium was substituted by pre-warmed Ca²⁺-free Hank's balanced salt solution before imaging. TG (0.5–1 μM) or 2.5 μM ionomycin was used to induce store depletion. Live cell imaging was performed at room temperature with × 60 oil lens on an inverted Nikon Eclipse Ti-E microscope customized with A1R-A1 confocal and motorized total internal reflection fluorescence (TIRF) modules using argon-ion (405 and 488 nm) and helium-neon (543 nm) or diode (561 nm) as laser sources. In some experiments, a Zeiss LSM 700 confocal system equipped with a × 100 oil lens (numerical aperture=1.45) was used to obtain images with higher resolution. Image analysis was performed using the NIS-Elements software (Nikon) or Image J (NIH).

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Intracellular PpIX Distribution in Macrophages

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THP-1-derived macrophages were incubated with serum-free RPMI 1640 medium containing 2 μM PpIX for 0.5, 1 and 4 h, followed by adding 1 μM PKH67 and 1 μM Hoechst 33342 for 5 min. After washing twice with phosphate-buffered saline (PBS), PpIX distribution was studied by a Zeiss LSM 700 confocal system (Zeiss, Germany).

Cao Z., Zhang T., Sun X., Liu M., Shen Z., Li B., Zhao X., Jin H., Zhang Z, & Tian Y. (2019). Membrane-permeabilized sonodynamic therapy enhances drug delivery into macrophages. PLoS ONE, 14(6), e0217511.

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Multimodal Microscopy for Cellular Signaling

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To image cAMP levels, the FRET-sensor H187 was excited at 405 nm and fluorescence was measured at 480 ± 10 nm and 530 ± 10 nm in xy-mode on a Zeiss LSM700 confocal system. To image NO levels, DAR-4M was AM-loaded into cells (10 µM DAR-4M AM, for 10 min), and its fluorescence was excited at 514 nm and emission detected at 580 ± 20 nm on a Leica TCS NT system.⁴⁷ (link) To image Ca²⁺ waves, Fluo3 was AM-loaded into myocytes (5 µM Fluo3, for 10 min), and its fluorescence was excited at 488 nm and detected >520 nm on a Leica TCS NT confocal system in xt mode. To measure pH_i, myocytes were AM-loaded with cSNARF1 (10 µM, for 10 min), and fluorescence was excited at 530 nm and detected simultaneously at 580 ± 10 nm and 640 ± 10 nm on an inverted Olympus microscope with an Orca 05G CCD Camera (Hamamatsu, Japan) and Optosplit (Cairn Research, UK).

Richards M.A., Simon J.N., Ma R., Loonat A.A., Crabtree M.J., Paterson D.J., Fahlman R.P., Casadei B., Fliegel L, & Swietach P. (2019). Nitric oxide modulates cardiomyocyte pH control through a biphasic effect on sodium/hydrogen exchanger-1. Cardiovascular Research, 116(12), 1958-1971.

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Immunostaining of Cardiomyocytes

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NRVMs were prepared for staining by means of Cardiomyocyte Immunocytochemistry Kit (Thermo Fisher Scientific) following manufacturer’s instruction. Briefly, NRVMs were fixed with Fixative Solution for 30 min, permeabilized with Permeabilization Solution S for 15 min at room temperature and then blocked with Blocking Solution for 30 min. Mitochondria were stained using anti-TOM20 antibody (Santa Cruz; 1:500, rabbit) and sarcomeres were stained using anti-α-sarcomeric actinin antibody (Sigma; 1:500, mouse) over-night at 4°C. The day after, samples were incubated with Alexa Fluor 594 conjugated anti-rabbit (Life Technologies, 1:250) and Alexa Fluor 488 conjugated anti-mouse (Life Technologies, 1:250) for 1h at room temperature. Coverslips were mounted using NucBlue™ Fixed Cell ReadyProbes™ Reagent with DAPI to stain nuclei (Invitrogen). Images were collected at Zeiss LSM 700 confocal system equipped with a PlanApo 40x/1.2 oil objective, and an Argon laser used to excite the fluorophores at the appropriate wavelength. Images were collected at 2048×2048 pixels per image with a 100 Hz acquisition rate. Images were analyzed using the Fiji distribution of the Java-based image processing program ImageJ [38 (link)]. The experiments were performed both in normoxia and after 6h of anoxia followed by 30 min of reoxygenation.

Antonucci S., Di Sante M., Sileikyte J., Deveraux J., Bauer T., Bround M.J., Menabò R., Paillard M., Alanova P., Carraro M., Ovize M., Molkentin J.D., Cohen M., Forte M.A., Bernardi P., Di Lisa F, & Murphy E. (2019). A novel class of cardioprotective small-molecule PTP inhibitors. Pharmacological research, 151, 104548.

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